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Cell nuclear transfer

a cell nuclear and transfer method technology, applied in the field of cell nuclear transfer in mammals, can solve the problems of high cost, high cost, time-consuming work, etc., and achieve the effect of low buoyant densities of porcine oocytes, high cost, and high cos

Active Publication Date: 2009-05-07
AARHUS UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The present invention relates to a method of cell nuclear transfer for creating genetically modified or transgenic non-human mammals. The method involves establishing an oocyte with a modified zona pellucida, separating the oocyte into parts to obtain a nucleus and cytoplasts, fusing the cytoplasts with a donor cell or cell nucleus, and obtaining a reconstructed embryo. The embryo can then be activated, cultured, and transferred to a host mammal to develop into a genetically modified fetus. The invention also includes a method for cryopreserving pig embryos and a genetically modified non-human embryo. The technical effects of the invention include improved efficiency and accuracy in creating genetically modified or transgenic non-human mammals.

Problems solved by technology

Micromanipulation, however, has proven to have several disadvantages for example the need for expensive equipment, the need for highly skilled personnel and time-consuming work.
However, a number of technical problems made HMC application in pig more demanding than originally supposed.
One of the problems encountered relates to low buoyant densities of porcine oocytes, both Zona intact (ZI) and especially zona-free (ZF) porcine oocytes.
Furthermore, the surface of the oocytes is sticky and it is hard to avoid their attachment to each other when zona is removed.
Moreover, ZF porcine oocytes are very fragile and it is difficult to bisect them in the way as described for bovine oocytes.
Recently, the HMC technique was, however with low efficiency, applied in porcine nuclear transfer, using genetically modified somatic cells, fibroblasts, as donor cells resulting in the production of genetically modified cloned blastocysts (Kragh et al.
An obstacle to producing genetically modified animals by nuclear transfer methods at a large scale is the inability of cryopreserving pig oocytes and embryos using methods applied to other species.

Method used

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Examples

Experimental program
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Effect test

example 1

[0160]Differences in developmental competence between sow (2.5 years, 170 Kg in weight) derived oocytes and gilt (5.5˜6 months, 75 Kg in weight) derived oocytes were investigated through ZF and ZI PA after 44 h in vitro maturation. Four combined groups were investigated in 3 identical replicates: (1) ZF oocytes from sows (2) ZI oocytes from sows (3) ZF oocytes from gilts (4) ZI oocytes from gilts. For ZF activation, a single DC pulse of 0.85 KV / cm for 80 μs was applied, while a single 1.25 KV / cm pulse was used to activate ZI oocytes. Following 7 days culture as described above, the percentage of blastocysts per activated embryo was determined.

[0161]The in vitro developmental competence of parthenogenetically activated oocytes derived from either sows or gilts was investigated. As shown in Table 1, the blastocyst rates of parthenogenetically activated oocytes from sows were significantly higher than those from gilts, either after ZF or ZI PA.

TABLE 1Blastocyst development of Day 7 par...

example 2

[0163]The feasibility of modified porcine HMC was investigated in 6 identical replicates, with IVF and in parallel ZF PA as controls. The more competent sow oocytes (according to Example 1) were used in Example 2. Seven days after reconstruction and / or activation, the number of blastocysts per reconstructed embryo and total cell numbers of randomly selected blastocysts were determined.

[0164]More than 90% of oocyte fragments derived from morphologically intact oocytes could be recovered for HMC after the trisection. In average, 37 embryos could be reconstructed out of 100 matured oocytes. The developmental competence of all sources of porcine embryos is shown in Table 2. On Day 7, the development of reconstructed embryos to the blastocyst stage was 17±4% with mean cell number of 46±5, while the blastocyst rates for IVF, and ZF PA were 30±6% and 47±4% (n=243, 170, 97) respectively.

TABLE 2In vitro development of embryosproduced by HMC, IVF and ZF PANo. ofblastocystMean cellEmbryoembryo...

example 3

[0167]Vitrification of hand-made cloned porcine blastocysts produced from delipated in vitro matured oocytes.

[0168]Recently a noninvasive procedure was published for delipation of porcine embryos with centrifugation but without subsequent micromanipulation (Esaki et al. 2004 Biol Reprod. 71, 432-6).

[0169]Cryopreservation of embryos / blastocysts was carried out by vitrification using Cryotop (Kitazato Supply Co, Fujinomiya Japan) as described previously (Kuwayama et al. 2005a; 2005b). At the time of vitrification, embryos / blastocysts were transferred into equilibration solution (ES) consisting of 7.5% (V / V) ethylene glycol (EG) and 7.5% dimethylsulfoxide (DMSO) in TCM199 supplemented with 20% synthetic serum substitute (SSS) at 39° C. for 5 to 15 min. After an initial shrinkage, embryos regained their original volume. 4˜6 embryos / blastocysts were transferred into 20 ul drop of vitrification solution (VS) consisting of 15% (V / V) EG and 15% (DMSO) and 0.5M sucrose dissolved in TCM199 su...

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Abstract

The present invention discloses methods for cell nuclear transfer that comprise for example modification of zona pellucida of an oocyte, and / or sectioning of oocytes into several parts. The present invention also discloses methods for producing a genetically modified non-human mammal. Genetically modified non-human mammals obtainable by the disclosed methods are also within the scope of the present invention. Disclosed are also methods for cryopreservation of cells.

Description

FIELD OF INVENTION[0001]The present invention relates to a method of cell nuclear transfer in mammals, and to genetically modified mammals obtained or genetically modified animals that can be obtained by the method. Furthermore the present invention relates to a method of vitrifying oocytes, zygotes, embryos including blastocysts.BACKGROUND OF INVENTION[0002]The ability to genetically modify donor cells and using them for nuclear transfer provides a tool for the production of genetically modified animals which may be used for example as disease models for the study of serious human diseases and drug testing.[0003]Traditional cell nuclear transfer techniques involve two steps of micromanipulation. A first step involves the enucleation of a mature oocyte, and a second step encompasses the transfer of a donor nucleus. Micromanipulation, however, has proven to have several disadvantages for example the need for expensive equipment, the need for highly skilled personnel and time-consumin...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): A01K67/027A01K67/00C12N15/873C12N15/877
CPCA01K2217/05C12N15/8778C12N15/877C12N15/85C12N5/06C12N5/00
Inventor DU, YUTAOBOLUND, LARS AXELVAJTA, GABORKRAGH, PETER MICHAEL
Owner AARHUS UNIV
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